Previously we established culture conditions for clonal expansion of cells in low calcium medium, which lead to the expression of mesenchymal markers (epithelial-mesenchymal transformation, EMT), and re-epithelialization in normal calcium medium, leading to partial regain of ductal and acinar markers (mesenchymal-epithelial transformation, MET). Cells expanded in low calcium medium and replated in Matrigel formed 3D structures that expressed some but not all ductal markers, and some, but not all acinar markers. We continued our studies in the areas as presented below. Comparison of SGSCs to established immortalized human salivary gland cell lines (HSG and HSY) Previously, we obtained HSG and HSY cell lines from Roberto Weigert, NIDCR in order to compare the phenotype of the SGSCs to lines known to express a salivary phenotype. In particular, we wanted to determine the efficacy of re-epithelialization of cells grown in low calcium medium, when switched to high calcium medium. In monolayer cultures, SGSC in low calcium media expressed Pan Cytokeratin (epithelial marker), Vimentin (mesenchymal marker), Cytokeratin 19 (ductal marker) and Aquaporin 5 (acinar marker). Surprisingly, although there was a clear change in morphology in high calcium media, fewer cells expressed these markers. HSG and HSY did not express these markers well. Each single-colony derived strains were also analyzed. The expression pattern was similar among the cell strains. Expression of putative stem cell markers by SGSCs Recent publications have suggested that salivary stem cells, obtained by ligating the duct to induce injury and thus, proliferation of a putative stem cell, express alpha6beta1 integrin and c-kit. We analyzed polyclonal and monoclonal cultures of SGSCs grown first in low calcium medium and the switched into high calcium medium for CD49f (alpha6), CD29 (beta1), and c-kit. The results show that alpha6beta1 is expressed at high levels when the calcium levels are increased, and that c-kit was expressed at only low levels. The same results were found in polyclonal and monoclonal cultures. SGSCs exhibit branching morphology by 3D culture with Matrigel and FGF10, and enhanced AQP5 mRNA expression SGSC previously grown in low calcium medium form 3D structures when cultured with Matrigel in high calcium medium, however, there is a great deal of lot-to-lot variation and on the company from which we purchased the Matrigel, and we explored this in more detail. We have now used more consistent lots (GFR, growth factor reduced Matrigel from Trevigen and BD;GRE, growth factor-enriched Matrigel from BD Biosciences). SGSCs switched from low calcium to high calcium medium formed small 3D structures when cultured with GFR, and formed duct and bud like structures when cultured with BD Matrigel. In addition, medium that contained 500ng/mL FGF10 induced more branching of the structure. SGSC transplanted with Matrigel and scaffold expressed amylase protein SGSCs grown in low calcium medium were transplanted into immunocompromised mice subcutaneously and into damaged salivary glands. SGSCs were mixed with Matrigel and directly injected, or the mixture of SGSCs and Matrigel was soaked into PGA/PLLA sponge and transplanted and harvested at 4weeks or 8weeks. When directly injected subcutaneously and into salivary glands, the cells formed cyst-like structures, indicative of fluid transport into the interior of the duct, although we were not able to capture this fluid for analysis. In the wall of the cyst like structures, the cells formed duct- and acinar- like structures but expressed only a ductal marker, Cytokeratin 19. In the PGA/PLLA sponge, the cells formed small acinar and duct like structures consistently spread throughout the transplant. These structures expressed a ductal marker (CK19) and an acinar marker (Amylase). HSG and HSY cells transplanted under the same conditions formed a carcinoma-like tissue and lacked organization into ductal or acinar-like structures, but did express both ductal and acinar markers. Based on these results, it is apparent that better ways are needed to encourage MET to form functional ductal and acinar elements in the appropriate architecture.